Electrochemical detection has been employed in liquid chromatography and in capillary electrophoresis (CE). It has been demonstrated that electrochemical detection is very sensitive and can measure 10.sup.-16 to 10.sup.-19 moles of sample with typical detection volumes from nL to pL (Ewing, A. G.; Mesaros, J. M.; Gavin, P. F., Electrochemical Detection in Microcolumn Separations, Anal. Chem., 66, 527A-536A, (1994); Voegel, P. D.; Baldwin, R. P., Electrochemical Detection with Copper Electrodes in Liquid Chromatography and Capillary Electrophoresis, American Laboratory, 28(2), 39-45, (1996)). Electrochemical methods have also been used to detect DNA (Shigenaga, M. K.; Park, J.-W.; Cundy, K. C.; Gimeno, C. J.; Ames, B. N., In Vivo Oxidative DNA Damage: Measurement of 8-hydroxy-2'-deoxyguanosine in DNA and Urine by High-Performance Liquid Chromatography with Electrochemical Detection, Methods in Enzymol., 186, 521-530, (1990); Takenaka, S.; Uto, H.; Knodo, H.; Ihara, T.; Takagi, M., Electrochemically Active DNA Probes-Detection of Target DNA Sequences at Femtomole Level by High-Performance Liquid Chromatography with Electrochemical Detection, Anal. Biochem., 218, 436-443, (1994); Johnston, D. H.; Glasglow, D. C.; Thorp, H. H., Electrochemical Measurement of the Solvent Accessibility of Nucleobases Using Electron Transfer Between DNA and Metal Complexes, J. Am. Chem. Soc., 117, 8933-8938, (1995)), single cells (Olefirowicz, T. M.; Ewing, A. G., Capillary Electrophoresis in 2 and 5 .mu.M Diameter Capillaries: Application to Cytoplasmic Analysis, Anal. Chem., 62, 1872-1876, (1990); Pihel, K.; Hsieh, S.; Jorgenson, J. W.; Wightman, R. M., Electrochemical Detection of Histamine and 5-Hydroxytryptamine at Isolated Mast Cells, Anal. Chem., 67, 4514-4521, (1995)), and even single molecules (Fan, F.-R. F.; Bard, A. J., Electrochemical Detection of Single Molecules, Science, 267, 871-874, (1995)). The operation of these electrochemical detectors is typically based on the use of three electrodes called the working, counter, and reference electrodes. There are three configurations which have been used to detect CE separations: on-column (Huang, X.; Pang, T.-K. J.; Gordon, M. J.; Zare, R. N., On-Column Conductivity Detector for Capillary Zone Electrophoresis, Anal. Chem., 59, 2747-2749, (1987), where the electrodes of the detector are placed within the capillary; end-column (Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G., End-Column Detection for Capillary Zone Electrophoresis, Anal. Chem., 63, 189-192, (1991); Chen, M.-C.; Huang, H.-J., An Electrochemical Cell for End-Column Amperometric Detection in Capillary Electrophoresis, Anal. Chem., 67, 4010-4014, (1995)), where the electrodes are placed directly at the end of the separation capillary; and off-column (Olefirowicz, T. M.; Ewing, A. G., Capillary Electrophoresis in 2 and 5 .mu.M Diameter Capillaries: Application to Cytoplasmic Analysis, Anal. Chem., 62, 1872-1876, (1990); O'Shea, T. J.; Greenhagen, R. D.; Lunte, S. M.; Lunte, C. E.; Smyth, M. R.; Radzik, D. M.; Watanabe, N., Capillary Electrophoresis with Electrochemical Detection Employing an On-Column Nafion Joint, J. Chromatogr., 593, 305-312, (1992); Wu, D.; Regnier, F. E.; Linhares, M. C., Electrophoretically Mediated Micro-Assay of Alkaline Phosphatase using Electrochemical and Spectrophotometric Detection in Capillary Electrophoresis, J. Chromatogr. B, 657, 357-363, (1994)), where the electrodes are electrically isolated from the electrophoresis voltage by a grounded porous glass tube. On-column electrochemical detection of CE separations has been performed by fixing two platinum wires through diametrically opposed holes drilled by a laser in a capillary tube. This structure is very difficult to manufacture and align, and the placement of the detection electrodes within the high voltage region of the separation column is problematic. In this format, one is trying to detect small currents or voltages while applying many kV to the separation column. The mechanical instability and poor definition of the electrode alignment can lead to significant electrical pickup or fluctuation in the background, making the desired signal very difficult to detect. The presence of high voltage gradients and significant electrophoretic currents in the column near the electrodes can induce stray signals. The end-column and off-column detection formats are important because they minimize the influence of the electrophoresis voltage. In the end-column format, one wants to place the detection electrodes as close to the end of the electrophoresis channel as possible so the detection is performed as close to ground potential as possible. This is very difficult to do with conventional manufacturing techniques. The electrodes must be placed with micron precision at the end of the capillary. Any error in the placement will cause loss of analyte signal if the electrodes are too far from the opening or high voltage pick up if the electrodes are placed within the separation column. Furthermore, fluctuations in electrode placement or electrode-electrode gap can cause severe fluctuations in the background signal producing noise. Typically, one must use micromanipulators and a microscope to assemble the detector. Furthermore, the engineering of the electrical isolation by connection of the separation and detection capillary tubes with a grounded porous glass tube in the off-column format is rather difficult to assemble and operate, and the junction can be mechanically unstable and poorly defined. In one case, although Slater and Watt (Slater, J. M.; Watt, E. J., On-chip Microbond Array Electrochemical Detector for use in Capillary Electrophoresis Analyst, 1994, 119, 2303-2307) photolithographically fabricated electrodes on a substrate, because they did not make a fully integrated separation and detection device, they were forced to use said undesirable junctions to couple their detector to a conventional cylindrical capillary.
There is a need for a microfabricated capillary electrophoresis chip with integral thin film electrochemical detector and electrophoresis leads which can be easily connected to associated electrical electrophoresis and detector apparatus.